Molecular surveillance of influenza A virus in Saudi Arabia: whole-genome sequencing and metagenomic approaches.

Outbreaks of influenza A viruses are generally seasonal and cause annual epidemics worldwide. Due to their frequent reassortment and evolution, annual surveillance is of paramount importance to guide vaccine strategies. The aim of this study was to explore the molecular epidemiology of influenza A virus and nasopharyngeal microbiota composition in infected patients in Saudi Arabia. A total of 103 nasopharyngeal samples from 2015 and 12 samples from 2022 were collected from patients positive for influenza A. Sequencing of influenza A as well as metatranscriptomic analysis of the nasopharyngeal microbiota was conducted using Oxford Nanopore sequencing. Phylogenetic analysis of hemagglutinin, neuraminidase segments, and concatenated influenza A genomes was performed using MEGA7. Whole-genome sequencing analysis revealed changing clades of influenza A virus: from 6B.1 in 2015 to 5a.2a in 2022. One sample containing the antiviral resistance-mediating mutation S247N toward oseltamivir and zanamivir was found. Phylogenetic analysis showed the clustering of influenza A strains with the corresponding vaccine strains in each period, thus suggesting vaccine effectiveness. Principal component analysis and alpha diversity revealed the absence of a relationship between hospital admission status, age, or gender of infected patients and the nasopharyngeal microbial composition, except for the infecting clade 5a.2a. The opportunistic pathogens Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were the most common species detected. The molecular epidemiology appears to be changing in Saudi Arabia after the COVID-19 pandemic. Antiviral resistance should be carefully monitored in future studies. In addition, the disease severity of patients as well as the composition of the nasopharyngeal microbiota in patients infected with different clades should also be assessed.IMPORTANCEIn this work, we have found that the clade of influenza A virus circulating in Riyadh, KSA, has changed over the last few years from 6B.1 to 5a.2a. Influenza strains clustered with the corresponding vaccine strains in our population, thus emphasizing vaccine effectiveness. Metatranscriptomic analysis showed no correlation between the nasopharyngeal microbiome and the clinical and/or demographic characteristics of infected patients. This is except for the 5a.2a strains isolated post-COVID-19 pandemic. The influenza virus is among the continuously evolving viruses that can cause severe respiratory infections. Continuous surveillance of its molecular diversity and the monitoring of anti-viral-resistant strains are thus of vital importance. Furthermore, exploring potential microbial markers and/or dysbiosis of the nasopharyngeal microbiota during infection could assist in the better management of patients in severe cases.

Enrichment of SARS-CoV-2 sequence from nasopharyngeal swabs whilst identifying the nasal microbiome.

Simultaneously characterising the genomic information of coronaviruses and the underlying nasal microbiome from a single clinical sample would help characterise infection and disease. Metatranscriptomic approaches can be used to sequence SARS-CoV-2 (and other coronaviruses) and identify mRNAs associated with active transcription in the nasal microbiome. However, given the large sequence background, unenriched metatranscriptomic approaches often do not sequence SARS-CoV-2 to sufficient read and coverage depth to obtain a consensus genome, especially with moderate and low viral loads from clinical samples. In this study, various enrichment methods were assessed to detect SARS-CoV-2, identify lineages and define the nasal microbiome. The methods were underpinned by Oxford Nanopore long-read sequencing and variations of sequence independent single primer amplification (SISPA). The utility of the method(s) was also validated on samples from patients infected seasonal coronaviruses. The feasibility of profiling the nasal microbiome using these enrichment methods was explored. The findings shed light on the performance of different enrichment strategies and their applicability in characterising the composition of the nasal microbiome.

Amplicon and Metagenomic Analysis of Middle East Respiratory Syndrome (MERS) Coronavirus and the Microbiome in Patients with Severe MERS.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional zoonotic spill over into humans. The severity of infection in humans is influenced by numerous factors, and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are coincident in the Middle East and thus a rapid way of sequencing MERS-CoV to derive genotype information for molecular epidemiology is needed. Additionally, complicating factors in MERS-CoV infections are coinfections that require clinical management. The ability to rapidly characterize these infections would be advantageous. To rapidly sequence MERS-CoV, an amplicon-based approach was developed and coupled to Oxford Nanopore long read length sequencing. This and a metagenomic approach were evaluated with clinical samples from patients with MERS. The data illustrated that whole-genome or near-whole-genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants, including deletion mutants. The metagenomic analysis provided information of the background microbiome. The advantage of this approach is that insertions and deletions can be identified, which are the major drivers of genotype change in coronaviruses. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in late 2012 in Saudi Arabia. The virus is a serious threat to people not only in the Middle East but also in the world and has been detected in over 27 countries. MERS-CoV is spreading in the Middle East and neighboring countries, and approximately 35% of reported patients with this virus have died. This is the most severe coronavirus infection so far described. Saudi Arabia is a destination for many millions of people in the world who visit for religious purposes (Umrah and Hajj), and so it is a very vulnerable area, which imposes unique challenges for effective control of this epidemic. The significance of our study is that clinical samples from patients with MERS were used for rapid in-depth sequencing and metagenomic analysis using long read length sequencing.

Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.